US11299545B2 - Anti-HLA-C antibodies and uses thereof - Google Patents

Anti-HLA-C antibodies and uses thereof Download PDF

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US11299545B2
US11299545B2 US16/839,179 US202016839179A US11299545B2 US 11299545 B2 US11299545 B2 US 11299545B2 US 202016839179 A US202016839179 A US 202016839179A US 11299545 B2 US11299545 B2 US 11299545B2
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variable region
chain variable
antigen
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Ryan Eberwine
Anne Fourie
Paul H. Kim
Carl L. Manthey
Hong Zhou
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Janssen Biotech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This invention relates to monoclonal anti-HLA-C antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies, and methods of using the antibodies to treat diseases including psoriasis, are also provided.
  • This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “JBI6065USNPSEQLIST.TXT” and a creation date of Mar. 28, 2019 and having a size of 126 kb.
  • the sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
  • autoimmune diseases afflict 1 in 20 persons (Hayter and Cook, Autoimmune Rev. 11(10):754-65 (2012)).
  • the root cause of autoimmunity is emergence of dysregulated T cell clones reactive to self-antigens.
  • Auto-reactive T cells initiate cytokine-driven feed-forward loops resulting in inflammation (e.g., leukocyte recruitment and edema), which when chronic, leads to tissue destruction and loss of function.
  • Antibodies that neutralize specific cytokines are useful to interrupt the feed-forward loops and minimize inflammation (Wolf and Ang, Immunol. Allergy Clin. North Am. 37(2):283-99 (2017); Her and Kavanaugh, J. Allergy Clin. Immunol. 137(1):19-27 (2016)).
  • these agents require chronic treatment, as they do not address the initial root cause of the disease. Therefore, therapies that target the development and activation of auto-reactive T cells and not the cytokines are needed to address the root cause of disease versus targeting the downstream inflammation.
  • T cells recognize antigens by a heterodimeric surface receptor consisting of an ⁇ and a ⁇ chain and known as the T cell receptor (TCR).
  • TCR T cell receptor
  • the ⁇ and ⁇ chain genes are subject to extensive somatic recombination in the thymus. Recombination during thymic T cell development gives rise to tremendous diversity in TCR genes expressed within an individual (Laydon et al., Philos. Trans. R. Soc. Long. B Biol. Sci. 370(1675):20140291 (2015)). Each TCR has distinct antigen recognition capability.
  • the antigens are short peptides bound and displayed by one of six major histocompatibility complex (MHC) proteins of the host.
  • MHC major histocompatibility complex
  • humans express three class I MHC genes (i.e., HLA-A, HLA-B, or HLA-C) and three class II MHC genes (i.e., HLA-DR, HLA-DP, or HLA-DQ) (Trowsdale, Immunol. Lett. 137(1-2):1-8 (2011)).
  • MHC genes are among the most polymorphic genes in the genome and there are >1000 alleles of each MHC gene in the human population encoding hundreds of allomorphs (protein isoforms).
  • Each MHC allomorph has a unique peptide binding surface and binds a unique repertoire of self and foreign peptides.
  • TCRs expressed by mature T cells are “restricted” to one of the self MHC allomorphs, meaning that a T cell can only be activated by a specific peptide-MHC complex.
  • HLA-C*06:02 allele Inheritance of the HLA-C*06:02 allele is the strongest genetic determinant of who will develop the autoimmune disease called psoriasis (Nair et al., Am. J. Hum. Genet. 78(5):827-51 (2006); Elder et al., J. Invest. Dermatol. 130(5):1213-26 (2010)).
  • a large majority ( ⁇ 80%) of individuals experiencing the guttate form of psoriasis carry the HLA-C*06:02 allele (Asumalahti et al., J. Invest. Dermatol. 120(4):627-32 (2003)), while about 50% of individuals with the more common plaque psoriasis carry this allele compared with about 10-15% of healthy control individuals.
  • HLA-C*06:02 One copy of the HLA-C*06:02 allele carries a ⁇ 10% lifetime risk, which is 2.5 ⁇ higher in homozygotes (Gudjonsson et al., Br. J. Dermatol. 148(2):233-5 (2003)), and individuals that are HLA-C*06:02 positive develop psoriasis at an earlier age and with greater severity (Gudjonsson et al., J. Invest. Dermatol. 126(4):740-5 (2006)).
  • the risk of psoriasis in HLA-C*06:02 individuals is epistatic with a catalytically more active variant of ERAP1 (Strange et al., N. Genet. 42(11):985-90 (2010)), the primary endopeptidase required to customize peptides for class I MHC loading.
  • Peptides derived from keratin-17, LL37, and ADAMTSL5 have been shown to be part of the repertoire of peptides that HLA-Cw6 may bind, and LL37 peptide-bound HLA-Cw6 tetramers have been used to identify cognate T cells in psoriasis patients. Meanwhile keratin-17 and peroxiredoxin-2-peptides activate T cells to a significantly greater frequency in HLA-C*06:02 positive psoriasis patients versus those that are negative.
  • Antibodies to HLA-C that block the development and activation of auto-reactive T cells could be efficacious in addressing the root problem of psoriasis.
  • the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind HLA-C, optionally, HLA-Cw6.
  • isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17, or a light chain variable region having a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or 18.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises:
  • the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric.
  • the isolated monoclonal antibody or antigen-binding fragment thereof is human or humanized.
  • the isolated monoclonal antibody or antigen-binding blocks development and activation of T cells, through binding and inhibition of antigen presentation by HLA-Cw6.
  • isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.
  • vectors comprising the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.
  • host cells comprising the vectors comprising the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.
  • a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
  • the methods comprise administering to the subject the pharmaceutical compositions of the invention.
  • the autoimmune disease can be selected from, but not limited to, psoriasis, plaque psoriasis, guttate psoriasis, and psoriatic arthritis.
  • kits for inhibiting activation of auto-reactive T cells in a subject in need thereof comprise administering to the subject the pharmaceutical compositions of the invention, wherein administration of the pharmaceutical composition inhibits activation of the auto-reactive T cells in the subject.
  • TCR T cell receptor
  • a peptide-bound HLA-C e.g., HLA-Cw6
  • HLA-C HLA-Cw6
  • the methods comprise contacting the cell with an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, wherein the monoclonal antibody or antigen-binding fragment thereof of the invention binds the peptide-bound HLA-C and inhibits the functional binding of the TCR.
  • the cell is contacted in vitro.
  • the cell is contacted in vivo.
  • the methods comprise culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof and recovering the antibody or antigen-binding fragment thereof from the cell or culture.
  • kits for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the methods comprise combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • FIG. 1 shows a graph demonstrating the inhibition of HLA-Cw6/ADAMTSL5-Abu dependent CD69 up-regulation on a T cell line by anti-HLA-C antibodies.
  • FIG. 2 shows a graph demonstrating the inhibition of HLA-Cw6/TRAT dependent CD69 up-regulation on T cell line by anti-HLA-C antibodies.
  • FIGS. 3A-3C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B130.001 Anti-HLA-C antibody.
  • FIGS. 4A-4C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B175.001 Anti-HLA-C antibody.
  • FIGS. 5A-5C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B228.001 Anti-HLA-C antibody.
  • FIGS. 6A-6C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B229.001 Anti-HLA-C antibody.
  • FIGS. 7A-7C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B230.001 Anti-HLA-C antibody.
  • FIGS. 8A-8C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B233.001 Anti-HLA-C antibody.
  • FIGS. 9A-9C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B237.001 Anti-HLA-C antibody.
  • FIGS. 10A-10C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B238.001 Anti-HLA-C antibody.
  • FIGS. 11A-11C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B188.001 Anti-HLA-C antibody.
  • FIGS. 12A-12C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the CW6B123.001 Anti-HLA-C antibody.
  • FIGS. 13A-13C show graphs demonstrating LabScreen Class I MHC allele binding profiles for the control CNTO9412 (huIgG4_PAA isotype) antibody.
  • any numerical values such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.
  • nucleic acids or polypeptide sequences e.g., anti-HLA-C antibodies and polynucleotides that encode them, HLA-C polypeptides and HLA-C polynucleotides that encode them
  • sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • the terms “inhibit,” “inhibiting,” and “inhibition,” mean to decrease an activity, response, condition, disease or other biological parameter. This can include, but is not limited to, complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between, as compared to native or control levels.
  • an antibody of the invention can inhibit the activation of an auto-reactive T cell by binding HLA-C (e.g., HLA-Cw6.
  • the invention generally relates to isolated anti-HLA-C antibodies (e.g., HLA-Cw6 antibodies), nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies.
  • Methods of making the antibodies, and methods of using the antibodies to treat diseases including autoimmune diseases (e.g., psoriasis) are also provided.
  • the antibodies of the invention possess one or more desirable functional properties, including but not limited to high-affinity binding to HLA-C, high specificity to HLA-C, the ability to inhibit activation of auto-reactive T cells, the ability to inhibit the functional binding of a TCR to a peptide-bound HLA-Cw6, and the ability to treat or prevent an autoimmune disorder in a subject in need thereof.
  • the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind HLA-C (e.g., HLA-Cw6).
  • HLA-C e.g., HLA-Cw6
  • antibody is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
  • the antibodies of the invention can be of any of the five major classes or corresponding sub-classes.
  • the antibodies of the invention are IgG1, IgG2, IgG3 or IgG4.
  • Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
  • the antibodies of the invention can contain a kappa or lambda light chain constant domain.
  • the antibodies of the invention include heavy and/or light chain constant regions from rat or human antibodies.
  • antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3).
  • the light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.
  • an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to HLA-Cw6 is substantially free of antibodies that do not bind to HLA-Cw6). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies of the invention can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods.
  • the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
  • the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab′, a F(ab′)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antibody fragment such as, for example, a diabody, a Fab,
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.
  • the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain.
  • the antigen-binding fragment comprises Fab and F(ab′).
  • single-chain antibody refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids.
  • single domain antibody refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • humanized antibody refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.
  • the first epitope is located on HLA-Cw6 and the second epitope is located on other autoimmune associated surface antigens.
  • HLA-Cw6 refers to a MHC class I heavy chain receptor.
  • HLA-Cw6 is one of the serotypes of the HLA-C class I heavy chain receptors.
  • the HLA-C receptor is a heterodimer consisting of a HLA-C mature gene product and the b2-microglobulin. The mature C chain is anchored in the cell membrane.
  • HLA-C are expressed in nearly all cells and serve to present small peptides to the immune system, which acts to determine if the small peptide is a self or non-self peptide.
  • HLA-C is a locus on chromosome 6, which encodes for a large number of HLA-C alleles that are Class-I-MHC receptors.
  • HLA-C proteins include, but are not limited to, HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw6, HLA-Cw7, HLA-Cw8, HLA-Cw12, HLA-Cw14, HLA-Cw15, HLA-Cw-16, HLA-Cw17, and HLA-Cw18.
  • An exemplary amino acid sequence of a human HLA-Cw6 is represented by GenBank Accession No. Q29963.
  • an antibody that “specifically binds to HLA-Cw6” refers to an antibody that binds to a HLA-Cw6, preferably a human HLA-Cw6, with a KD of 1 ⁇ 10 ⁇ 7 M or less, preferably 1 ⁇ 10 ⁇ 8 M or less, more preferably 5 ⁇ 10 ⁇ 9 M or less, 1 ⁇ 10 ⁇ 9 M or less, 5 ⁇ 10 ⁇ 10 M or less, or 1 ⁇ 10 ⁇ 10 M or less.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2, a HCDR3, a light chain complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3, having the polypeptide sequences of:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the polypeptide sequence of:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the polypeptide sequence of:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17, or a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or 18.
  • the isolated monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region having the polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99/a identical to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or 18, respectively.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the invention, comprising:
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:37, 38, 39, 64, 65, and 66, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:40, 41, 42, 67, 68, and 69, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:43, 44, 45, 70, 71, and 72, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:6.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:46, 47, 48, 73, 74, and 75, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7; and a light chain variable region having the polypeptide sequence of SEQ ID NO:8.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:49, 50, 51, 76, 77, and 78, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9; and a light chain variable region having the polypeptide sequence of SEQ ID NO:10.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:52, 53, 54, 79, 80, and 81, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11; and a light chain variable region having the polypeptide sequence of SEQ ID NO:12.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:55, 56, 57, 82, 83, and 84, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13; and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:58, 59, 60, 85, 86, and 87, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15; and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:61, 62, 63, 88, 89, and 90, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:18.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:17; and a light chain variable region having the polypeptide sequence of SEQ ID NO:18.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:91, 92, 93, 118, 119, and 120, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:94, 95, 96, 121, 122, and 123, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:97, 98, 99, 124, 125, and 126, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:6.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:100, 101, 102, 127, 128, and 129, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7; and a light chain variable region having the polypeptide sequence of SEQ ID NO:8.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:103, 104, 105, 130, 131, and 132, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9; and a light chain variable region having the polypeptide sequence of SEQ ID NO:10.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:106, 107, 108, 133, 134, and 135, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11; and a light chain variable region having the polypeptide sequence of SEQ ID NO:12.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:109, 110, 111, 136, 137, and 138, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13; and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:112, 113, 114, 139, 140, and 141, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15; and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:115, 116, 117, 142, 143, and 144, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:18.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:17; and a light chain variable region having the polypeptide sequence of SEQ ID NO:18.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:145, 146, 147, 172, 173, and 174, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:148, 149, 150, 175, 176, and 177, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:151, 152, 153, 178, 179, and 180, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:6.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:154, 155, 156, 181, 182, and 183, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7; and a light chain variable region having the polypeptide sequence of SEQ ID NO:8.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:157, 158, 159, 184, 185, and 186, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9; and a light chain variable region having the polypeptide sequence of SEQ ID NO:10.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:160, 161, 162, 187, 188, and 189, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11; and a light chain variable region having the polypeptide sequence of SEQ ID NO:12.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:163, 164, 165, 190, 191, and 192, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13; and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:166, 167, 168, 193, 194, and 195, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15; and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOs:169, 170, 171, 196, 197, and 198, respectively.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:18.
  • the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:17; and a light chain variable region having the polypeptide sequence of SEQ ID NO:18.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is chimeric.
  • the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is human or humanized.
  • the invention in another general aspect, relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the coding sequence of a protein can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding monoclonal antibodies or antigen-binding fragments thereof of the invention can be altered without changing the amino acid sequences of the proteins.
  • the invention in another general aspect, relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an antibody or antigen-binding fragment thereof in the cell.
  • Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the invention.
  • the invention in another general aspect, relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen-binding fragments thereof of the invention.
  • the host cells are E. coli TG1 or BL21 cells (for expression of, e.g., an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells or HEK293 cells (for expression of, e.g., a full-length IgG antibody).
  • the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
  • the invention in another general aspect, relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce a monoclonal antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant).
  • Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.
  • the invention in another general aspect, relates to a pharmaceutical composition, comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutical composition as used herein means a product comprising an antibody of the invention together with a pharmaceutically acceptable carrier.
  • Antibodies of the invention and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.
  • the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used in the invention.
  • compositions of the invention are known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions).
  • additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents.
  • One or more pharmaceutically acceptable carrier can be used in formulating the pharmaceutical compositions of the invention.
  • the pharmaceutical composition is a liquid formulation.
  • a preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation comprising water.
  • the liquid formulation can comprise a solution, a suspension, an emulsion, a microemulsion, a gel, and the like.
  • An aqueous formulation typically comprises at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w/w of water.
  • the pharmaceutical composition can be formulated as an injectable which can be injected, for example, via an injection device (e.g., a syringe or an infusion pump).
  • the injection can be delivered subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously, for example.
  • the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use.
  • Solid dosage forms can include tablets, such as compressed tablets, and/or coated tablets, and capsules (e.g., hard or soft gelatin capsules).
  • the pharmaceutical composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.
  • the dosage forms can be immediate release, in which case they can comprise a water-soluble or dispersible carrier, or they can be delayed release, sustained release, or modified release, in which case they can comprise water-insoluble polymers that regulate the rate of dissolution of the dosage form in the gastrointestinal tract or under the skin.
  • the pharmaceutical composition can be delivered intranasally, intrabuccally, or sublingually.
  • the pH in an aqueous formulation can be between pH 3 and pH 10.
  • the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.
  • the pharmaceutical composition comprises a buffer.
  • buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof.
  • the buffer can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific buffers constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises a preservative.
  • preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof.
  • the preservative can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.
  • Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises an isotonic agent.
  • an isotonic agent such as sodium chloride
  • an amino acid such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine
  • an alditol such as glycerol, 1,2-propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol
  • polyethyleneglycol e.g. PEG400
  • Another example of an isotonic agent includes a sugar.
  • Non-limiting examples of sugars can be mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose.
  • Another example of an isotonic agent is a sugar alcohol, wherein the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one —OH group.
  • Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the isotonic agent can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.
  • Pharmaceutical compositions comprising each one of these specific isotonic agents constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises a chelating agent.
  • chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.
  • the chelating agent can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.
  • Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises a stabilizer.
  • stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.
  • the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy-/hydroxycellulose and derivates thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulphur-containing substances such as monothioglycerol), or thioglycolic acid.
  • the stabilizer can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants.
  • surfactant refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic) part.
  • the surfactant can, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants.
  • the surfactant can be present individually or in the aggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the invention.
  • the pharmaceutical composition comprises one or more protease inhibitors, such as, e.g., EDTA, and/or benzamidine hydrochloric acid (HCl).
  • the protease inhibitor can be present individually or in the aggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml.
  • Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.
  • the invention in another general aspect, relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining a monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • the invention in another general aspect, relates to a method of treating or preventing an autoimmune disease in a subject in need thereof.
  • the method comprises administering to the subject an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds HLA-Cw6 or a pharmaceutical composition of the invention.
  • the HLA-Cw6 monoclonal antibody or antigen-binding fragment thereof is capable of binding HLA-Cw6 on cells in the subject.
  • Binding of the anti-HLA-Cw6 monoclonal antibody or antigen-binding fragment thereof to a peptide-bound HLA-Cw6 on the cell surface can allow for inhibition of activation of the auto-reactive T cell, and/or can allow for the inhibition of functional binding of the peptide-bound HLA-Cw6 to a T cell receptor (TCR).
  • TCR T cell receptor
  • the anti-HLA-Cw6 monoclonal antibody can, for example, form a bispecific antibody with another monoclonal antibody or antigen-binding fragment that can also inhibit activation of an auto-reactive T cell in the subject.
  • the functional activity of antibodies and antigen-binding fragments thereof that bind HLA-Cw6 can be characterized by methods known in the art and as described herein.
  • Methods for characterizing antibodies and antigen-binding fragments thereof that bind HLA-Cw6 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; binding assays to detect the binding of antibodies to HLA-Cw6 on T cells by FACS.
  • the methods for characterizing antibodies and antigen-binding fragments thereof that bind HLA-Cw6 include those described below.
  • the invention in another general aspect, relates to a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen binding fragment thereof that specifically binds HLA-Cw6 or a pharmaceutical composition of the invention.
  • the autoimmune disease can, for example, be selected from but not limited to, psoriasis, plaque psoriasis, guttate psoriasis, and psoriatic arthritis.
  • the pharmaceutical composition comprises a therapeutically effective amount of an anti-HLA-Cw6 antibody or antigen-binding fragment thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount means an amount of the anti-HLA-Cw6 antibody or antigen-binding fragment thereof that modulates an immune response in a subject in need thereof.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to an autoimmune disease or disorder (e.g., psoriasis), which is not necessarily discernible in the subject, but can be discernible in the subject.
  • the terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as an autoimmune disorder (e.g., psoriasis).
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • compositions used in the treatment or prevention of an autoimmune disease can be used in combination with another treatment including, but not limited to, a chemotherapy, an anti-IL-23 mAb, an anti-TNF-alpha mAb, an anti-IL17A mAb, other autoimmune disease drugs, an antibody-drug conjugate (ADC), or a targeted therapy.
  • Anti-HLA-Cw6 antibodies can be used to construct bispecific antibodies with partner mAbs against IL-23, TNF-alpha, IL17A, and/or other T cell surface antigens to treat autoimmune diseases that express both HLA-Cw6 and the specific T cell surface antigen.
  • a first therapy e.g., a composition described herein
  • a first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to
  • This invention provides the following non-limiting embodiments.
  • Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and a HCDR3, the light chain variable region comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of:
  • Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-3, comprising a heavy chain variable region having a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or 17, or a light chain variable region having a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or 18.
  • Embodiment 5 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-4, comprising:
  • Embodiment 6 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-5, wherein the antibody or antigen-binding fragment thereof is chimeric.
  • Embodiment 7 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-6, wherein the antibody or antigen-binding fragment thereof is human or humanized.
  • Embodiment 8 is the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-8, wherein the antibody or antigen-binding fragment thereof blocks development and activation of T cells, through binding and inhibition of antigen presentation by HLA-Cw6.
  • Embodiment 9 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-8.
  • Embodiment 10 is a vector comprising the isolated nucleic acid of embodiment 9.
  • Embodiment 11 is a host cell comprising the vector of embodiment 10.
  • Embodiment 12 is a pharmaceutical composition, comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-8 and a pharmaceutically acceptable carrier.
  • Embodiment 13 is a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 12.
  • Embodiment 14 is the method of embodiment 13, wherein the autoimmune disease is selected from the group consisting of psoriasis, plaque psoriasis, guttate psoriasis, and psoriatic arthritis.
  • Embodiment 15 is a method of inhibiting the activation of an auto-reactive T cell in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 12.
  • Embodiment 16 is a method of inhibiting the functional binding of a T cell receptor (TCR) to a peptide-bound HLA-Cw6 complex in a HLA-Cw6 expressing cell, the method comprising contacting the cell with an isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-8, wherein the monoclonal antibody or antigen-binding fragment thereof binds the peptide-bound HLA-Cw6 and inhibits functional binding the TCR to the peptide-bound HLA-Cw6 complex.
  • TCR T cell receptor
  • Embodiment 17 is the method of embodiment 16, wherein the cell is contacted in vitro.
  • Embodiment 18 is the method of embodiment 16, wherein the cell is contacted in vivo.
  • Embodiment 19 is a method of producing the monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-8, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions to produce the monoclonal antibody or antigen-binding fragment thereof and recovering the antibody or antigen-binding fragment thereof from the cell or culture.
  • Embodiment 20 is a method of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any one of embodiments 1-8, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Recombinant HLA-Cw6 with peptides were expressed in both mammalian and bacterial expression systems.
  • recombinant A2PW23 (rhHLA-A2) (SEQ ID NO:199), MHGW2 (rhHLA-G5) (SEQ ID NO:200) and MO1W6 ( ⁇ 2-microglobulin) (SEQ ID NO:201) antigens were used to identify antibodies with high selectivity to HLA-Cw6.
  • Cell lines stably expressing target antigens were generated using standard cloning, expression and clonal selection methods.
  • 96-Well Nunc F96 MaxiSorp plates were coated with a 50 ⁇ l/well of a 1 ⁇ g/ml solution (PBS) containing a 1:1 mixture of CW6W32 (rhHLA-Cw6/TRAT) (SEQ ID NO:202) and CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)) antigens for a minimum of 2.5 hours prior to aspiration of the coating solution.
  • the plates were blocked with 200 ⁇ l/well blocking solution (0.4% BSA/PBS) for 3-4 days at 4° C.
  • the plates were washed 3 ⁇ 300 ⁇ l/well with 1 ⁇ PBST and 50 ⁇ l/well of culture supernatants, including control samples, were added and the plate incubated for 1 hour at RT.
  • the plates were washed, as described above, and 50 ⁇ l/well of a goat anti-rat IgG Fc-HRP (diluted 1:10K) (Jackson Immunoresearch; West Grove, Pa., #112-036-071) secondary detection reagent, prepared in blocking buffer, was added to each well.
  • the plate was incubated for 30 minutes at RT and then washed as described above.
  • the hits (880) arising from the primary screen of hybridoma fusions were screened against additional antigens to confirm binding selectivity and specificity.
  • TMB Thermo #34022
  • the hits (880) arising from the primary screen of hybridoma fusions were screened against additional antigens to confirm binding selectivity and specificity.
  • the coating solutions were discarded, and the plates blocked with 150 ⁇ l/well blocking buffer (20% FBS and 0.18% NaN 3 in PBS) for 30 minutes at RT.
  • the blocking solution was discarded, and 50 ⁇ l/well of supernatant was added to the assay plates and incubated for 1 hour at RT before washing the plates with 3 ⁇ 300 ⁇ l/well PBS.
  • the secondary SulfoTag labeled anti-rat IgG detection antibody (Meso Scale Discovery (MSD); Rockville, Md., #R32AH-1), prepared as a 0.5 mg/ml solution, was then added to the plates at 50 ⁇ l/well and incubated for 1 hour at RT before washing the plates as described above.
  • the plates then received 150 ⁇ l of a 1 ⁇ MSD Read Buffer T (surfactant free) (MSD #R92TD-1), and the plates were read on an MSD Sector Plate Reader.
  • the coated plates were washed with 75 ⁇ l 1 ⁇ PBST and 60 ⁇ l/well of blocking buffer (0.4% BSA/PBS) were added to all plates. The plates were sealed and incubated overnight at 4° C. On the following day, the plates were washed, as described above, and 10 ⁇ l/well of harvested supernatants from the single B cell cultures were incubated in the plates for 2 hours at RT. The plates were washed and 15 ⁇ l/well of a 1 pg/ml goat SulfoTag anti-mouse secondary detection antibody (MSD #R32AC-5) was added to the assay plate, and the plate was incubated for 1 hour at RT. The plates were washed and 35 ⁇ l/well of 1 ⁇ MSD Read Buffer T (MSD #R92TC-1) added and the plates read on a MSD Sector Imager 6000.
  • MSD #R92TC-1 1 ⁇ MSD Read Buffer T
  • K562 parent and K562 cell lines stably expressing rhHLA-Cw6 (K562-rhHLA-Cw6) (SEQ ID NO:211) and rhHLA-B7 (K562-rhHLA-B7) (SEQ ID NO:213) were seeded into 384-well MSD High Bind assay plates (MSD #L21XB-4) at 12000 cells/well density. After a 1-hour incubation at 37° C. and 5% CO 2 in an incubator, the plate was aspirated, 50 ⁇ l of Starting Block blocking buffer (Thermo Fisher #37542) was added to each well, and the plate was incubated further for 30 minutes at RT.
  • Starting Block blocking buffer Thermo Fisher #37542
  • the blocking buffer was aspirated, 20 ⁇ l of prepared test antibodies were added to appropriate wells, and the plate was incubated further for 1 hour at RT.
  • the plate was washed three times with 80 ⁇ l PBS, and then each well received 20 ⁇ l/well of a 1 ⁇ g/ml SulfoTag labeled secondary detection antibody solution. After a 1-hour incubation at RT, the plate was washed three times with 80 ⁇ l PBS, and 50 ⁇ l/well of a 1 ⁇ MSD Read Buffer T (surfactant free) (MSD #R92TD-1) was added and the plate was read on an MSD Sector 600 Plate Reader.
  • MSD #R92TD-1 1 ⁇ MSD Read Buffer T (surfactant free)
  • a Jurkat T cell clone stably expressing both CD8 (SEQ ID NO:216 and SEQ ID NO:217) and TCR-ADAMTSL5 (SEQ ID NO:214 and SEQ ID NO:215) (clone E11) (Jurkat-rhCD8/rhTCR-ADAMTSL5) are stimulated with a LCL.721.221 clone stably expressing rhHLA-Cw6 (LCL.721.221-rhHLA-Cw6) (SEQ ID NO:211) to induce an IL-2 cytokine release response.
  • the cytokine release response is dependent on the ADAM-Abu peptide loading on the LCL.721.221-rhHLA-Cw6 due to the specificity of the TCR-ADAMTLS5 response from the Jurkat cell clone.
  • LCL.721.221-rhHLA-Cw6 were loaded with 0.2 mg/ml ADAM-Abu (SEQ ID NO:218) peptide for 1 hour in a humidified incubator set at 37° C. and supplemented with 5% CO 2 .
  • the peptide-loaded LCL.721.221-rhHLA-Cw6 cells were plated into a 384-well assay plate at 20 ⁇ l/well and incubated with 20 ⁇ l of 4 ⁇ test antibodies for 1 hour in a humidified incubator set at 37° C. and supplemented with 5% CO 2 .
  • the Jurkat-rhCD8 + /rhTCR-ADAMTSL5 cells were then added to the assay plate at 40 ⁇ l/well, and the assay plate was incubated for 16-20 hours in a humidified incubator set at 37° C. and supplemented with 5% CO 2 .
  • 20 ⁇ l/well were harvested and analyzed using a 384-well MSD IL-2 Cytokine assay kit (MSD #K211AHB-2) using the manufacturers recommended protocol.
  • Jurkat T cell clones stably expressing TCR-ADAMTLS5 (clone E11) (SEQ ID NO:214 and SEQ ID NO:215) (Jurkat-rhTCR-ADAMTSL5) and TCR-TRAT (SEQ ID NO:225 and SEQ ID NO:226) (clone C5) (Jurkat-rhTCR-TRAT) were stimulated with K562 cell line stably expressing HLA-Cw6 (clone 16) (K562-rhHLA-Cw6) (SEQ ID NO:211) in the presence of cognate peptide to activate the Jurkat T cell clones as measured by up-regulation of CD69 using flow cytometry.
  • the appropriate Jurkat T cell clones were added at 50 ⁇ l/well (50K cells/well) to the assay plates containing the cognate peptide loaded K562-HLA-Cw6 cells, and the plates were incubated at 37° C. for 14-15 hours. After the treatment period was complete, the cells were transferred to a 96-well round-bottom plate (Costar #3799), and the plate was centrifuged at 1300 RPM for 5 minutes at RT.
  • the cells were then incubated for 30 minutes on ice with 50 ⁇ l/well with a staining cocktail, prepared with FACS Stain Buffer, containing Cell Trace CFSE (ThermoFisher #C34554) and BV421 anti-human CD69 antibody (mIgG1, clone FN50, BD Pharmingen #562883).
  • FACS Stain Buffer containing Cell Trace CFSE (ThermoFisher #C34554) and BV421 anti-human CD69 antibody (mIgG1, clone FN50, BD Pharmingen #562883).
  • FACS Stain Buffer containing Cell Trace CFSE (ThermoFisher #C34554) and BV421 anti-human CD69 antibody (mIgG1, clone FN50, BD Pharmingen #562883).
  • 150 ⁇ l/well of FACS Stain Buffer was added to each well, and the plate spun down
  • the LABScreen (One Lambda) assay investigates antibody binding to a panel of 97 Class I HLA alleles (HLA-A, -B, and -C) using a bead-based fluorescent flow technology (Luminex 100; Luminex; Austin, Tex.).
  • Luminex 100 Luminex 100; Luminex; Austin, Tex.
  • 5 ⁇ l of the LABScreen (One Lambda #LS1A04) beads were incubated with 20 ⁇ l of each test antibody (10 ⁇ g/ml) for 30 minutes in the dark at RT with gentle shaking. After the incubation was complete, 150 ⁇ l of the supplied 1 ⁇ wash buffer was added to each well, and the plate was centrifuged at 1300 ⁇ g for 5 minutes at RT.
  • the wash solution was removed by flicking, and the plates were washed one more time.
  • a 1 ⁇ PE-conjugated anti-human IgG (One Lambda #LSAB-2) solution was prepared and 100 ⁇ l/well added to the assay plate.
  • the plate was incubated for 30 minutes in the dark at RT with gentle shaking.
  • the beads were then washed twice as described above, and then 80 ⁇ l/well of 1 ⁇ PBS was added to each well.
  • the plate was analyzed on a Luminex 100 instrument, and the data was analyzed using the Fusion 3.0 Software (One Lambda).
  • Binding of anti-HLA-Cw6 mAbs to multiple HLA-Cw6 antigens was measured on a ProteOn XPR36 protein interaction array system (Bio-Rad; Hercules, Calif.) using GLC sensor chips (Bio-Rad #176-5011). Goat anti-human Fc antibody (Jackson ImmunoResearch (Jackson #109-005-098)) was conjugated to the chip by standard amine coupling in the horizontal orientation for the subsequent capture of anti-HLA-Cw6 mAbs (“ligands”) in the vertical orientation. Anti-HLA-Cw6 mAbs were captured at ligand densities ranging from 100 to 200 response units.
  • Binding to each HLA-Cw6 antigen (“analyte”) was measured by flowing five antigen concentrations (100 nM diluted down to 1.23 nM in a 3-fold concentration series) in the horizontal orientation over captured mAbs, with a sixth analyte channel containing only buffer (PBST—phosphate-buffered saline supplemented with 0.005% Tween20, pH 7). All analyses were conducted in PBST at 100 ⁇ L/min flow rate with association and dissociation times of 180 and 900 seconds, respectively. Chip surface regeneration (release of captured ligand) was achieved by 2 short pulses of 0.85% phosphoric acid (18s contact time at 100 ⁇ L/min).
  • Analyte raw data was double referenced by subtraction of the blank surface response at “interspots” (to correct for refractive index changes and any non-specific interactions with the chip surface) and of the buffer blank response (to correct for any baseline drift resulting from mAb dissociation over time). Referenced data were globally fit to a 1:1 simple Langmuir binding model. Kinetic parameters were calculated for each mAb/antigen combination or interaction using Bio-Rad ProteOn Manager software (version 3.1.0.6) with parameter (kon, koff, Rmax) scope and type set to “Grouped” and “Fitted,” respectively.
  • CM4 sensor chips (GE Healthcare #BR-1005-34) were prepared by coupling goat anti-human IgG Fc antibody (Jackson #109-005-098) to the chip surface using an amine coupling kit (GE Healthcare #BR-1000-50).
  • the running buffer for all kinetic measurements was 1 ⁇ HBSP+100 ⁇ g/ml BSA and all measurements were obtained at 25° C.
  • Single-cycle kinetic analysis was performed by immobilizing 75-225 RU of test antibodies followed by injections of antigens at three concentrations (3.7-100 nM) with a contact time of 60 seconds and dissociation time of 900 seconds at 60 ⁇ l/min flow rate. Resulting sensorgrams were fitted using the 1:1 kinetic model with global k a , k d and Rmax and RI set to 0.
  • a CFM 2 (Wasatch Microfluidics; Salt Lake City, Utah) was used to create a microarray of 96 mAbs. It draws forty-eight 70- ⁇ l plugs of sample from a 96-well microplate into a fluidic manifold which focuses the solutions into an array of 48 micro flow cells on the surface of the SPR substrate (a G-COOH coated prism from Ssens, NL) and cycles the solutions back and forth at 60 ⁇ l/min.
  • a 96-well microplate was prepared with 100 ⁇ l of each mAb at 30 ⁇ g/ml in MES coupling buffer pH 4.5 and loaded into bay 2 of the CFM.
  • a second plate of freshly mixed activating reagents (150 ⁇ l 0.4 M EDC and 150 ⁇ l 0.1 M sulfo-NHS in a total of 5 ml of MES coupling buffer pH 4.5) was loaded into bay 1.
  • the CFM was then primed with system buffer (PBS+0.01% T20).
  • the anti-HLA-DR4 mAb plate contained 89 mAbs.
  • the activating reagents were cycled over the surface for 7 minutes and followed immediately by the first set of mAbs (top half of the mAb plate) and cycled for 15 minutes. Without undocking, the spots were rinsed with the system buffer.
  • the CFM Since the CFM prints 48 solutions at a time, it needs to address the surface twice to create the full array of 89 mAbs. After the first print, the CFM was paused to load fresh activation reagents, and the same cycle of 7-minute activation and 15-minute coupling was repeated for the second half of the mAb plate. The printed prism was then loaded into the SPRi reader (MX96, IBIS Technologies; Netherlands), which uses a single flow cell and autosampler configured to address the array with back-and-forth cycled injections of 80 ⁇ l per analyte. Once loaded, 1 M ethanolamine (GE Healthcare) was injected across the chip for 15 minutes to quench the excess reactive esters.
  • SPRi reader MX96, IBIS Technologies; Netherlands
  • reaction spots i.e., the 96-ligand array
  • interstitial reference spots two local reference spots per reaction spot.
  • CW6W3.ECO.PP.002/.003 rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)
  • All SPRi experiments were conducted in a 96 ⁇ 96 analyte-on-ligand format.
  • the injection valve and pepsin/protease XIII column and their related connecting tubing were inside a cooling box maintained at 16° C.
  • the second switching valve, C8 column and their related connecting stainless steel tubing were inside another chilled circulating box maintained at ⁇ 6° C.
  • Peptide identification was done through searching MS/MS data against the W6 sequence with Mascot.
  • the mass tolerance for the precursor and product ions were 7 ppm and 0.02 Da, respectively. 100% sequence coverage was achieved for CW6W3.
  • Hydrogen/Deuterium (H/D) exchange at each time point was performed in duplicates.
  • the exchange was quenched by adding 130 ⁇ L of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH was 2.5). Subsequently, the quenched samples were subjected to on column pepsin/protease XIII digestion and LC-MS analysis as described above. The mass spectra were recorded in MS only mode. Raw MS data was processed using HDX WorkBench, software for the analysis of H/D exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521). The deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form (t0).
  • OmniRats were immunized twice weekly for a total of 12 immunization boosts by following a Repetitive Immunizations Multiple Sites (RIMS) and final cell immunization boost protocol.
  • Recombinant human CW6W3.ECO.PP.002/.003 rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)
  • CW6W32 rhHLA-Cw6/TRAT
  • Sera was collected and assessed for circulating IgG specific antibodies to rhHLA-Cw6 and titers were determined via a solid phase ELISA with antigen coated directly on the plate. Lymph nodes were harvested for B lymphocytes fusion and the supernatants from the hybridomas were screened for binding to target antigens.
  • IP Intraperitoneal injection
  • 30 day protocol IP/RIMS with mixture of CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)) and CW6W32 (rhHLA-Cw6/TRAT) (SEQ ID NO:202) for a total of 8 injections over the course of the immunization protocol.
  • B cells were enriched from splenocytes by using AutoMACS MicroBeads to deplete T cells and IgM+ B cells. A portion of cells were fused for hybridoma generation. The remainder of B cells were stained with bt-HLA-CW6 antigen, anti-mouse IgG (Fcy-specific)-AlexaFluor647, anti-human Kappa-PE/Cy7 and sorted by FACS Aria III.
  • Single antigen-specific B cells (positive for msFc, huKappa, and HLA-CW6) were deposited into a 384-well tissue culture plate containing media (Medium E+20% Jurkat conditioned media+0.1 ⁇ g/ml msMegaCD40L (Enzo)+5 ⁇ g/ml msCpG ODN1826+25 ng/ml msIL-2 (Peprotech)+10 ng/ml msIL-6 (R&D Systems)+17 ng/ml msIL-10 (R&D Systems)+10 ⁇ g/ml LPS 026:B6 (Sigma)+1 ⁇ g/ml PWM (Sigma)+1800 iMEF feeder cells/well) and a single cell was deposited per well.
  • media Medium E+20% Jurkat conditioned media+0.1 ⁇ g/ml msMegaCD40L (Enzo)+5 ⁇ g/ml msCpG ODN18
  • Sorted cells were incubated for 4 days in 90 ⁇ l media and culture supernatants were screened by MSD for presence of mouse IgG antibodies against CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)) protein and various cell lines (parental, HLA-CW6, HLA-A, and HLA-B-overexpressing).
  • the supernatants from the OMT hybridomas were tested for binding to multiple recombinant antigens to select for antibodies selective to HLA-Cw6 by standard ELISA.
  • the six target antigens that were coated on the ELISA plate were (1) CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)), (2) CW6W32 (rhHLA-Cw6/TRAT) (SEQ ID NO:202), (3) CW6W36 (rhHLA-Cw6/ADAMTSL5) (SEQ ID NO:204), (4) A2PW23 (rhHLA-A2) (SEQ ID NO:199), (5) MHGW2 (rhHLA-G5) (SEQ ID NO:200), and (6) M01W6 ( ⁇ 2-microglobulin (SEQ ID NO:201).
  • the supernatants from the single B cells were screened by coating MSD plates with CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)) and detecting bound mouse IgG with a SulfoTag anti-mouse IgG secondary antibody.
  • the supernatants were also screened for binding to K562 cell lines stably expressing human HLA-Cw6 (K562-rhHLA-Cw6) (SEQ ID NO:211), HLA-A1 (K562-rhHLA-A1) (SEQ ID NO:212) and HLA-B7 (K562-rhHLA-B7) (SEQ ID NO:213); parent K562 cells were included to exclude non-specific binders.
  • Antibodies were identified from the supernatants of hybridoma fusion clones, from the OMT OmniRat immunization, and B cell sorting from Ablexis mice immunizations. From the characterization experiments discussed below, nine (9) unique antibodies that represent 8 antagonist and 1 non-antagonist anti-HLA-Cw6 antibodies with differing combined properties of epitope binding group, antagonist potency, binding selectivity and affinity were identified. In the sections below, the results from each in vitro characterization study is discussed for all 9 antibodies.
  • Hybridoma supernatants from the OMT OmniRat immunizations were initially screened in a 232 ⁇ 96-Well ELISA to identify clones expressing anti-HLA-Cw6 antibodies that bind CW6W32 (rhHLA-Cw6/TRAT) (SEQ ID NO:202) and CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)), the immunogens used for immunization. A total of 880 hits were identified from the primary screen.
  • a confirmatory ELISA screen was performed against multiple recombinant antigens and multiple cell lines stably expressing HLA-I molecules.
  • the antigens in the confirmatory screen were: (1) CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)), (2) CW6W32 (rhHLA-Cw6RAT) (SEQ ID NO:202), (3) CW6W36 (rhHLA-Cw6/ADAMTSL5) (SEQ ID NO:204), (4) A2PW23 (rhHLA-A2) (SEQ ID NO: 199), (5) MHGW2 (rhHLA-G5) (SEQ ID NO:200), and (6) M01CW6 (rh ⁇ 2-macroglobulin) (SEQ ID NO:201).
  • the confirmatory cell binding screen was performed against K562 cell lines stably expressing rhHLA-Cw6 (K562-rhHLA-Cw6) (SEQ ID NO:211) and counter-screened against K562 cells stably expressing rhHLA-A (K562-rAHLA-A) (SEQ ID NO:212), and the K562 parent cell line.
  • K562 cell lines stably expressing rhHLA-Cw6 K562-rhHLA-Cw6
  • K562-rAHLA-A K562-rAHLA-A
  • CW6W32 (rhHLA- ELISA 21 matory PP.002/.003 Cw6/TRAT) (rhHLA-Cw6/ARF)
  • CW6W36 (rhHLA- (negative for CW6W32 Cw6/ADAMTLS5, and CW6W36) C6V) (negative for A2PW23 rhHLA-A, (rhHLA-A2) rhHLA-G5 MHGW2 and ⁇ 2- (rhHLA-G5) microglobulin)
  • Cell K562-rhHLA-Cw6 K562-rhHLA-A1 MSD 123 Binding K562 Parent TOTAL 208
  • an additional primary cell binding screen was performed on the 208 supernatant hits that included a K562 cell line expressing HLA-B7 (K562-rhHLA-B7) (SEQ ID NO:213) to increase the specificity of the hybridoma clone supernatant panel to HLA-Cw6.
  • K562-rhHLA-B7 K562-rhHLA-B7
  • SEQ ID NO:213 49 hybridoma clone supernatants were identified to have selective binding to K562-rhHLA-Cw6 (SEQ ID NO:211) but not to K562-rhHLA-B7 (SEQ ID NO:213) or K562 parent cell lines.
  • the expressed and purified monoclonal antibodies were screened for selective binding to K562-rhHLA-Cw6 cell lines but not to K562-rehHLA-B7 or K562 parent cell lines using the MSD Cell Binding assay format.
  • a total of 7 mAbs met criteria for selectivity in binding K562-rhHLA-Cw6 but not K562-rhHLA-B7 or parent K562 cell line (Table 7).
  • HLA-Cw6 selective hits from purified monoclonal antibodies derived from B cell sorting 1) HLA-B7 2) HLA-Cw6 3) HLA-Cw6 Specific Specific Selective (K562- (K562- rhHLA-Cw6 rhHLA- rhHLA- specific/K562- B7/K562 Cw6/K562 rhHLA-B7 mAb Source Parent) Parent) specific) CW6B228 Single B Cell 1.0 75 78 CW6B229 Single B Cell 1.0 18 18 CW6B230 Single B Cell 0.7 40 61 CW6B232 Single B Cell 1.2 15 13 CW6B233 Single B Cell 0.8 36 44 CW6B237 Single B Cell 0.9 14 16 CW6B238 Single B Cell 0.9 14 15 1) HLA-B7 Specific antibodies are those that have a ratio >5 of [K562-rhHLA-B7 binding signal/K562 parent binding signal] 2) HLA-Cw
  • the purified antibodies identified from the primary screening of OMT OmniRat hybridoma and Ablexis B cell sorting were characterized for functional activity in two separate, but orthogonal, assays: (1) inhibition of HLA-Cw6/ADAMTSL5-Abu dependent IL-2 release in a T cell line, and (2) inhibition of HLA-Cw6/ADAM-TSL5 and HLA-Cw6/TRAT dependent CD69 up-regulation in T cell lines.
  • the IL-2 inhibition functional screen was performed at 3 antibody concentrations to rank-order the antibodies based on % inhibition of IL-2 release. Briefly, LCL-721.221-rhHLA-Cw6 cells (clone 28) (SEQ ID NO:211), which were loaded with ADAMTSL5-Abu peptide (SEQ ID NO:218), were co-cultured with a Jurkat T cell clone expressing human TCR-ADAMTSL5 receptor (SEQ ID NO:214 and SEQ ID NO:215) (Jurkat-rhTCR-ADAMTSL5) (HLA-Cw6/ADAMTSL5-Abu peptide is a cognate ligand).
  • the seven antibodies with good efficacy in blocking IL-2 release were characterized in an orthogonal functional bioassay that measured the inhibition of HLA-Cw6/ADAM-Abu and HLA-Cw6/TRAT dependent Jurkat T cell activation as measured by CD69 upregulation.
  • K562 cells stably expressing rHLA-Cw6 were loaded with ADAM-abu, or TRAT, peptide, treated with anti-HLA-Cw6 antibodies, and co-cultured with Jurkat T cell lines stably expressing TCR-ADAMTLS5, or TCR-TRAT, respectively. After an overnight incubation, the HLA-Cw6/peptide dependent up-regulation of CD69 expression was monitored by flow cytometry.
  • All seven antagonist antibodies from the IL-2 inhibition assay show antibody concentration dependent blocking of CD69 up-regulation on Jurkat-TCR-ADAMTSL5 ( FIG. 1 ) and Jurkat-TCR-TRAT ( FIG. 2 ) T cell lines.
  • CW6B130 showed highest potency and CW6B237 the lowest potency.
  • Cw6B237 has the highest potency.
  • HLA-A, -B and -C Class I HLA alleles
  • Luminex 100 bead-based fluorescent flow technology
  • HLA FusionTM software version 4.0
  • CW6B237 and CW6B238 show the highest specificity to HLA-C alleles, followed by CW6B130, CW6B175 and CW6B188 which all have cross-reactivity to HLA-B46, and CW6B130 and CW6B175 showing additional cross-reactivity to HLA-B73. Sequence analysis of these HLA-I alleles show HLA-B73 is closely related to the HLA-C family, while HLA-B46 appears to be contained within the HLA-B family.
  • valine at position 76 that is conserved in HLA-C, HLA-B46 and HLA-B73 alleles but not in other HLA-B alleles, suggesting a role of this valine in the allelic specificity observed with these antibodies.
  • the remainder of the antibodies show cross-reactivity (qualitatively) to at least >10 non-HLA-C alleles, with CW6B238 having the highest cross-reactivity across the 97 class I MHC alleles tested ( FIGS. 3A-3C, 4A-4C, 5A-5C, 6A-6C, 7A-7C, 8A-8C, 9A-9C, 10A-10C, 11A-11C, 12A-12C, and 13A - 13 C).
  • the antibodies with highest specificity to HLA-C alleles show good antagonist efficacy but not the highest activity.
  • the highest antagonist efficacy belongs to CW6B130 and CW6B175, which shows, qualitatively, cross-reactivity to HLA-B46 and HLA-B73.
  • CW6B188 which has high HLA-C selectivity but binds HLA-B46 has no antagonist activity in the T cell activation studies.
  • Affinity measurements of anti-HLA-Cw6 antibodies were obtained by Surface Plasmon Resonance (SPR) methods using a ProteON XPR36 system, as already described, against rHLA-Cw6/ARFN (SEQ ID NO:205) (acidic peptide in refolded rHLA-Cw6, aka CW6W3.ECO.PP.002/.003) (SEQ ID NO:203), rHLA-Cw6/TRAT (basic peptide in single-chain expressed protein, aka CW6W32) (SEQ ID NO:202) and rHLA-Cw6/ADAMTSL5 (acidic peptide in single-chain expressed protein, aka CW6W36) (SEQ ID NO:204) antigens.
  • SPR Surface Plasmon Resonance
  • CW6B123 Pos n/a n/a n/a No/low Ctrl binding response Antigen-CW6W32 (rhHLA-Cw6/TRAT) AA ID Source ka (1/Ms) kd (1/s) KD (nM) Comments CW6B130 OMT 1.03E+07 2.72E ⁇ 03 0.27
  • Valid kinetic fit CW6B175 OMT n/a n/a n/a Binding confirmed, fit not valid CW6B228 Ablexis 5.98E+06 5.34E ⁇ 04 0.09
  • Valid kinetic fit CW6B229 Ablexis 1.67-1.75E+06 1.25-1.90E ⁇ 03 0.713-1.14 Valid kinetic fit.
  • Anti-HLA-Cw6 epitopes on target antigen (CW6W3.ECO.PP.002/.003 (rhHLA-Cw6 (SEQ ID NO:203)/ARF (SEQ ID NO:219)) was determined by classical competition (sandwich format) experiment using SPRi (SPR imaging) and CFM (continuous flow microfluidic, or microspotter) printing and analyzed using the IBIS method. There were 6 total bin groups identified using a panel of 94 anti-HLA-C antibodies, and the 9 identified antibodies fit into three distinct epitope groups. Four of them bin together with the positive/reference control antibody, CW6B 123, in Group 3; four bin together in Group 4 and one antibody, CW6B237, bins into Group 6 (Table 11)

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